Limonene was administered once a day for 4 days and the striatum was sampled 24 hours after last injection. The tissues were homogenized with 1x lysis buffer (RIPA buffer, Thermo Scientific, MA, USA) including 1x protease inhibitor (Thermo Scientific) and incubated on ice for 30 min, and centrifuged at 13,000 rpm for 150 min at 4°C. An equal amount of total protein (30 μg) was resolved on 12% sodium dodecyl sulfate polyacrylamide gel and then transferred to a PVDF membrane (Immobilon-P; pore size 0.45 μm, EMD Millipore, Burlington, MA, USA). The membranes were blocked for 1 h in 5% skim milk solution and incubated for overnight at 4°C with glutamate decarboxylase 67 (Gad67; 1:1000, Sigma Aldrich, USA) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:1000, EMD Millipore). The blots were then incubated with secondary antibody; goat anti-mouse IgG-horseradish peroxidase (HRP) (1:5000, Sigma Aldrich, St. Louis, MO, USA). Immunoreactive proteins were detected with an enhanced chemiluminescence Western blotting detection system. The relative density of the protein bands was scanned and quantified by ImageJ (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).
Glyceraldehyde 3 phosphate dehydrogenase gapdh
Manufactured by Merck Group
Sourced in United States, Germany, Japan
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is an enzyme that catalyzes the sixth step of glycolysis, the metabolic pathway that converts glucose into energy. GAPDH is involved in the oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (7 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
E cadherin, by BD (3 mentions)
Peroxidase conjugated anti mouse or anti rabbit igg, by Santa Cruz Biotechnology (3 mentions)
Occludin, by Thermo Fisher Scientific (2 mentions)
Ddk tag, by OriGene (2 mentions)
Reporter lysis buffer, by Promega (2 mentions)
Ve cadherin, by Thermo Fisher Scientific (2 mentions)
Tissuelyser 2, by Qiagen (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Versadoc imaging system, by Bio-Rad (2 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Iblot device, by Thermo Fisher Scientific (2 mentions)
Amersham imaging system, by GE Healthcare (2 mentions)
N cadherin, by BD (2 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Il 1β, by Santa Cruz Biotechnology (2 mentions)
72 protocols using glyceraldehyde 3 phosphate dehydrogenase gapdh
1
Limonene Effects on Striatal Gad67 Levels
2
Western Blot Analysis of Lipoma Cells
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For Western blot analysis, lipoma cells were seeded at a density of 10,000 cells/cm² in culture medium. Cells were incubated with different alpelisib and/or rapamycin concentrations for 24 h. Proteins were extracted and immunoblotting was performed as described elsewhere [4 (link)]. We used 10 µg protein per lane and incubated with primary antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA) and secondary antibodies (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA), according to Table 2 . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Merck KGaA, Darmstadt, Germany) was used as loading control.
3
Protein Extraction and Western Blot Analysis
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For protein extraction, the cells were lysed in cell lysis buffer (140 mM NaCl, 10 mM Tris-HCl, 1% Triton X-100, 1 mM ECTA and 1X protease inhibitor cocktail). The protein concentration was determined using the Bradford reagent method. A total of 25 µg protein was loaded into each lane of a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and subsequently blotted onto a polyvinylidene difluoride membrane. Following blocking with PBS with Tween-20 containing 5% nonfat dry milk at room temperature for 1 h, the membrane was incubated with primary antibodies directed against β1-integrin (1:200; cat. no. AB1952; EMD Millipore) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5,000; cat. no. 2118; Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. The membrane was then incubated with peroxidase-linked immunoglobulin G secondary antibodies (Thermo Fisher Scientific, Inc.). The proteins were visualized using an Enhanced Chemiluminescence Western Blotting Detection kit (GE Healthcare, Chicago, IL, USA) according to the manufacturer's protocol. The western blot analysis results were quantified using ImageJ software (the National Institutes of Health).
4
Western Blot Analysis of Mimecan and IGFALS
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Proteins (25 μg) in each sample were precipitated using the methanol:chloroform method, then boiled in 2X Laemmli buffer and loaded onto polyacrylamide gels. Following SDS-PAGE, the proteins were transferred onto PVDF membranes, which were then blocked in Tris-buffered saline (TBS) containing 0.1% [v/v] Tween and 3% [w/v] BSA. Membranes were incubated overnight at 4 °C with primary antibodies against mimecan (OGN, 1/4000 dilution, Invitrogen, CA, USA) and insulin-like growth factor-binding protein complex acid labile subunit (IGFALS, 1/1000 dilution, OriGene, Rockville, USA), diluted in blocking buffer, then washed extensively in TBS containing 0.1% [v/v] Tween before and after incubation for 1 h with HRP-conjugated secondary antibodies (1/20,000 dilution). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1/10,000 dilution, Merck) was used as a loading control. Immunodetection was achieved with ECL Classico substrate (Merck).
5
Western Blot Antibody Dilutions
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We used Hamlet 1 (1:500; Leica Microsystems, Wetzlar, Germany; Mueller et al., 2014 (link)), Romeo (1:1000; Abcam; Mueller et al., 2014 (link)), mitsugumin-53/TRIM72 (1:5000; a generous gift from Jianjie Ma, University of Medicine and Dentistry of New Jersey, Piscataway, NJ; Cai et al., 2009b (link)), c-Myc (1:500; Santa Cruz Biotechnology, Dallas, TX), β-tubulin (1:1000; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH: 1:5000; Merck, Millipore), OTOF-C12 (Santa Cruz Biotechnology), and anti-myoferlin 7D2 (Abcam; Leung et al., 2012 (link)).
6
Protein Detection and Quantification in Keratinocytes
7
Western Blot Analysis of Lung Proteins
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Western blot was performed as previously described18 (link). Briefly, half of the left lobe of mouse lungs were homogenized in 300 μl of lysis buffer (1× Reporter lysis buffer (Promega, Madison, WI, USA), containing protease inhibitor (Roche, Basel, Switzerland)) using a TissueLyser II (Qiagen, Germantown, MD, USA). Supernatants were used for SDS-PAGE and western blots. Thirty micrograms of total protein were loaded on 10% SDS-PAGE gels, transferred onto PVDF membrane and probed with primary antibodies against DDK tag (Origene, Rockville, MD, USA), ZO-1, occludin, and VE-cadherin (Invitrogen, Rockford, IL, USA), β1-Na+, K+-ATPase (Millipore, Billerica, MA, USA), MRCKα (Santa Cruz, Dallas, TX, USA) and glyceraldehyde-3-phosphate dehydrogenase GAPDH (EMD Millipore, Burlington, MA, USA). Data were analyzed using the NIH Image J software.
8
Lung Protein Expression Analysis
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Western blot was performed using half of the left lung lobe that was homogenized in 300 μl of 1× Reporter lysis buffer (Promega, Madison, WI, USA), containing protease inhibitor (Roche, Basel, Switzerland) using a TissueLyser II (Qiagen, Germantown, MD, USA) as previously described14 (link). Following SDS-PAGE and transfer to PVDF membrane, blots were probed with primary antibodies against DDK tag (Origene, Rockville, MD, USA), ZO-1 (Invitrogen, Rockford, IL, USA), occludin (Invitrogen), VE-cadherin (Invitrogen, Rockford, IL, USA), and glyceraldehyde-3-phosphate dehydrogenase GAPDH (EMD Millipore, Burlington, MA, USA). Data were analyzed using the NIH Image J software.
9
Protein Expression Analysis in Rat Tissues
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After the rats were scarified, the gastrocnemius muscle and liver were quickly frozen in liquid nitrogen, and stored at −80 °C. The tissues were homogenized with the T-PER™ tissue protein extraction buffer (Pierce Biotechnology, Thermo-Fisher, Rockford, IL, USA) containing 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail (Calbiochem, Merck Millipore, Darmstadt, Germany). The protein samples in the supernatants were collected after centrifugation at 15,000× g at 4 °C for 10 min, and analyzed by Western blotting with primary antibodies detecting IRS-1 (1:1000, GeneTex, San Antonio, TX, USA), PI3-k p85 α (1:1000; Cell Signaling Technology, Beverly, MA, USA), SREBP1c, CD36 (1:1000; Novus Biologicals, Littleton, CO, USA), and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:20,000; Merck Millipore), and then detected with secondary antibodies (anti-rabbit or anti-mouse IgG HRP-conjugated). The blots were quantified by chemiluminescence with MiniChemi I system (Beijing Sage Creation Science, Beijing, China), and then normalizing with GAPDH. The relative protein intensity was expressed as folds of the content in the NC group.
10
Western Blot Analysis of Key Signaling Proteins
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The cells were lysed in a radioimmunoprecipitation assay buffer (50 mM Tris HCl, pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 5 mM EDTA, 1 mM EGTA, 5 mM DTT, 2 mM phenylmethylsulfonyl fluoride, and 10 μg/mL leupeptin) and incubated on ice for 30 min. After centrifugation, the supernatant was collected and subjected to Western blotting analysis. The antibodies used in this study were as follows: HSD3B (sc-30820 or sc-100466; Santa Cruz Biotechnology, TX, USA), STAT6 (Thermo), phospho-STAT6 (Tyr641; Thermo), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Millipore, MA, USA), β-actin (Sigma-Aldrich, St. Louis, MO, USA ), AKT (Santa Cruz Biotechnology), phospho-AKT (Ser473; Cell Signaling Technology, MA, USA), p-GSK3β (Ser9; Cell Signaling Technology, Danvers, MA, USA), and GSK3β (BD Biosciences, Franklin Lakes, NJ, USA).
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